Self-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for spermatogenesis. Worldwide, millions of men experience infertility or sub-fertility which may be due to impairment of SSC functions. Also, spermatogenesis is a classic stem cell-dependent process which can serve as a model for other tissue-specific stem cell systems. Thus, deciphering the mechanisms that regulate SSC fate decisions is of critical importance. Similar to other adult stem cell populations, SSC functions are controlled extrinsically by influence of a niche microenvironment and intrinsically via activation of specific molecular networks. Previous studies have determined that glial cell line-derived neurotrophic factor (Gdnf) is an essential growth factor regulating self-renewal of mammalian SSCs. The influence of growth factors on stem cell fate decisions is mediated via regulating specific intrinsic molecular networks, which are currently poorly understood in SSCs. We showed that Gdnf stimulates expression of the transcriptional repressor, inhibitor of differentiation 4 (Id4) in the Thy1+ germ cell population of mouse testes which is composed of SSCs and non-stem cell spermatogonia. Additionally, we determined that lack of Id4 expression in vivo disrupts normal spermatogenesis in mice resulting in depletion of the proliferating spermatogonia population. Furthermore, reduction of Id4 expression in wild-type SSCs abolishes their ability to self-renew in vitro. These observations indicate that Id4 is an essential intrinsic regulator of SSC fate decisions;however, its mechanism of action is unknown. Id4 is a member of the basic helix-loop-helix (bHLH) family of transcription regulators, but lacks a DNA binding domain. In other tissues, the Id family of proteins maintain progenitor cells in an undifferentiated state by binding to and repressing the activity of specific bHLH transcription factors that stimulate differentiation. In the mouse male germline, Id4 is the only Id family protein expressed by spermatogonia and the bHLH transcription factors neurogenin 3 (Ngn3) and spermatogenesis and oogenesis helix-loop-helix 1 (Sohlh1) are regulators of early spermatogonial differentiation. Interaction between Id4 and these bHLH transcription factors in SSCs has not been examined. Our central hypothesis is that balance between self-renewal and differentiation of mouse SSCs is controlled by an intrinsic molecular pathway in which Id4 promotes self-renewal by repressing the activity of specific bHLH transcription factors that induce spermatogonial differentiation such as Ngn3 and Sohlh1. This hypothesis will be tested by;1) determining whether Id4 has the singular capacity to control SSC self-renewal, 2) determining whether Ngn3 and Sohlh1 are regulators of SSC differentiation, and 3) determining whether Id4 represses the transcription factor activity of Ngn3 and Sohlh1. PUBLIC HEALTH RELEVANCE: Deciphering the role of Id4, Ngn3, and Sohlh1 in control of SSC fate decisions will enhance overall understanding of foundation processes regulating spermatogenesis and male fertility. This information may be useful in diagnosing and treating specific incidences of reproductive failure in human males. Also, this research will expand general knowledge of stem cell biology that may be applicable to other tissue-specific stem cell populations important for sustaining life.